Cytotoxic T lymphocytes and natural killer cells release granzymes and perforin from cytotoxic granules into the immune synapse to induce apoptosis of virally infected or tumor targets. Granzyme A and granzyme B induce distinct pathways of programmed cell death. Granzyme A activates a caspase-independent apoptotic pathway. Granzyme A induces mitochondrial damage characterized by superoxide anion generation and loss of the mitochondrial transmembrane potential without interfering with mitochondrial outer membrane permeability and also causes single-stranded DNA damage by activating two nucleases in the SET complex. The SET complex is normally associated with the endoplasmic reticulum, but rapidly translocates to the nucleus in response to superoxide anion. The goal of this proposal is to continue to unravel the mechanism of action of granzyme A, building upon the research accomplished during the previous funding periods. To initiate mitochondrial and nuclear damage, granzyme A enters the mitochondrial matrix and the nucleus. One goal of this proposal is to define the mechanisms by which granzyme A penetrates the membranes surrounding these organelles. Another goal is to identify additional physiologically relevant mitochondrial and nuclear substrates of granzyme A. A third goal will focus on the SET complex, the engine of granzyme A-mediated DNA damage. Preliminary data suggest that the SET complex contains the phosphatase PP2A and that PP2A regulates the nuclear translocation of the SET complex. This proposal will confirm that PP2A is in the SET complex and determine how granzyme A alters the SET complex and causes it to traffic to the nucleus. PUBLIC HEALTH RELEVANCE: Granzyme A, the most abundant of the killer cell death-inducing proteases, activates a novel cell death pathway that is important in the immune defense against viruses and cancer, especially those that have evolved ways to avoid caspase-mediated cell death. There is now evidence that this pathway may also be activated endogenously by ischemia and seizures in the brain and that mutations in the granzyme A-activated DNase TREX1 that render cells resistant to granzyme A are genetically linked to inflammatory neurological diseases and systemic lupus erythematosis. Understanding the mechanisms behind the granzyme A pathway should not only improve our understanding of how the immune system controls tumors and infections, it should also help us understand normal cellular pathways and the response to oxidative stress, which granzyme A hijacks to cause death, and potentially provide insight into autoimmunity and cell death in the brain. These insights may lead to new therapeutic approaches to treat the conditions that granzyme A targets (intracellular infection and cancer) or potentially for autoimmunity and neurological injury.